The vertebrate immune system can evolve antibodies capable of recognizing essentially any macromolecule with high affinity and specificity. Analyses of natural antibody sequences together with structural studies have been instrumental in revealing how antibodies work (Chothia et al., 1992, J. Mol. Biol., 227: 799-817; Kabat, 1982, Pharmacological Rev., 34: 23-38; Kabat, 1987, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.)). These studies have revealed that antigen recognition is primarily mediated by complementarity determining regions (CDRs) that are located at one end of the antibody variable domain and are connected by a β-sheet framework (Wu & Kabat, 1970, J. Exp. Med., 132: 211-250; Kabat & Wu, 1971, Annals New York Acad. Sci., 190: 382-393). The sequence diversity of natural antibodies shows that the CDRs are hypervariable in comparison with the framework, and it is the CDR sequences that determine the antigen specificity of a particular antibody (Jones et al., 1986, Nature, 321: 522-5; Amit et al., 1986, Science, 233: 747-53). These studies have also revealed that the natural sequence diversity at most CDR positions is not completely random, as biases for particular amino acids occur in both a site-specific manner and in terms of overall CDR composition (Davies & Cohen, 1996, Proc. Natl. Acad. Sci. USA, 93: 7-12; Kabat et al., 1977, J. Biol. Chem., 252: 6609-16; Zemlin et al., 2003, J. Mol. Biol., 334: 733-49; Mian et al., 1991, J. Mol. Biol., 217: 133-51; Padlan, 1994, Mol. Immunol., 31: 169-217).
The diversity of antibody variable domains is generated by several distinct genetic mechanisms (Zemlin et al., 2003; Bassing et al., 2002, Cell, 109: S45-55; Tonegawa, 1983, Nature, 302: 575-81; Padlan, 1997, Mol. Immunol., 34: 765-70; Wilson, 2000, J. Exp. Med., 191: 1881-94). Diversity is encoded in the collection of V genes in the germline and further generated through recombination of V, D and J segments in B cells. Recombination generates variability in amino acid content in all CDRs and variation in the length of the loop portion of CDR3. Finally, somatic hypermutation during the secondary immune response generates diversity throughout variable domains. While these genetic mechanisms allow sufficient diversity to generate a biologically competent immune repertoire, they also likely constrain natural diversity as compared to the level of diversity created through a purely random process.
Phage display is a powerful technique that has been utilized to identify novel antigen binding antibody variable domains. The ability to identify and isolate high affinity antibodies from a phage display library is important in isolating novel human antibodies for therapeutic use. Isolation of high affinity antibodies from a library is dependent on the size of the library, the efficiency of production in cells, and the diversity of the library. See, for e.g., Knappik et al., J. Mol. Biol. (1999), 296:57. The size of the library is decreased by inefficiency of production due to improper folding of the antibody or antigen binding protein and the presence of stop codons. Expression in bacterial cells can be inhibited if the antibody or antigen binding domain is not properly folded. In some cases, expression can be improved by mutating residues in turns at the surface of the variable/constant interface, or at selected CDR residues. (Deng et al., J. Biol. Chem. (1994), 269:9533, Ulrich et al., PNAS (1995), 92:11907-11911; Forsberg et al., J. Biol. Chem. (1997), 272:12430). The sequence of the framework region is important in providing for proper folding when antibody phage libraries are produced in bacterial cells. However, not all strategies provide a library of diverse but structurally stable antibody variable domains.
Understanding the structural constraints of diversified antibody variable domains may contribute to providing a library of diverse yet structurally stable antibody variable domains that can bind to a target antigen with high affinity. Such a library and the antibody variable domains isolated from the library are useful as novel antigen binding molecules for use, for example, therapeutically and for ease of production of antibodies or antigen binding fragments on a large scale.